A kind of high-flux cell Combined culture chip
Technical field
The invention belongs to cell biological medical domains, and in particular to a kind of to realize high-throughput Coculture core
Piece.
Background technology
Micro-fluidic chip has many advantages, such as that at low cost, equipment is small, highly integrated, sample consumption is few, is widely applied to thin
Born of the same parents' biological field.RESEARCH ON CELL-BIOLOGY is developed to and is interacted to various kinds of cell just to single cell and research
Observation and research.For complicated biological systems, need the simultaneous platform of various kinds of cell that could preferably simulate very
Cell micro-environment existing for reality.To between more comprehensively deep explorations cell complex network and mediate mutual between them
The signal path etc. of effect.Coculture is the more commonly used method that interacts between research cell and cell.
Conventional cell co-cultivation plays simultaneously though INTERACTION PROBLEMS between many cells can be solved under ex vivo situation
The effect of certain simulated in vivo environment, but it is current, and the micro-fluidic chip based on cell culture is limited to two kinds or four kinds of cells more
Combined culture or a kind of target cell are mixed with other various kinds of cell, the cell category that can be cultivated simultaneously on a kind of chip
Less, flux is small, efficiency is low, under this state, isolated cells phenotype with it is in vitro before differ greatly, limit to each thin in microenvironment
Born of the same parents' component is observed, if carrying out the sensitivity Detection of chemotherapeutics to cell under this state, can be limited to result judgement
Accuracy, can not true environment in more true analogue body.In addition, the culturing room for being currently used in the micro-fluidic chip of medicine sieve fills
Connection structure without microbridge one kind between circulation road and nutrient solution perfusion channel and drug infusion channel, mostly two relatively independent
Parallel channel limits the contact area in nutrient solution perfusion channel and drug infusion channel and cell culture chamber.
Invention content
The present invention relates to overcome the technological deficiency that the cell category that can be cultivated simultaneously on a kind of chip is less, flux is small
And a kind of high-flux cell Combined culture chip, including N group cultivation platforms, N >=1, multigroup rounded divergent shape of cultivation platform point
Cloth is sequentially communicated between N group cultivation platforms or between relatively independent or every group by microbridge, or selectivity two adjacent groups extremely
It is adjacent(N-1)It is connected to by microbridge between group;Every group of cultivation platform includes three-decker, from inside to outside sequentially connected internal layer battalion
Unit, middle layer biogum perfusion unit and okioplast culture unit is perfused in nutrient solution, is connected to by microbridge between three;Every group
Cultivation platform includes 1-10 cell culture unit, is connected to by microbridge between flanking cell culture unit.
The internal layer nutrient solution perfusion unit include sequentially connected inlet, flow channel for liquids, liquid cell, flow channel for liquids,
Liquid outlet;In order to increase the area of liquid cell, increases the storage volume of nutrient solution, nutrition, the liquid are persistently provided for cell
Pond uses triangular funne shape, the vertex of the liquid cell in multiple cultivation platforms to be directed toward the same center of circle.
Further, the cell culture unit includes cell import, and one end is closed, and the other end is connected by cell runner
Cell culture chamber;The other end of the culturing room connects cell outlet by cell runner;The culturing room is rectangular.
Further, the middle layer biogum perfusion unit is equipped with glue-feeder, colloid runner and gum outlet;The colloid
The place contacted with the cell culture chamber on runner is equipped with microbridge.
In order to increase the contact area of cell culture chamber and biogum perfusion unit and nutrition perfusion unit, the colloid
Runner include main stem and(N-1)Branch, main stem form the frame of entire biogum perfusion unit, are trained across entire cell
Unit is supported, head end and end are separately connected glue-feeder and gum outlet;Between each two cell culture unit, being used for will for branch
Adjacent cell culture unit is connected to by microbridge, and head end is connected to main stem, and end connects gum outlet.
In order to which the matrigel ensured in colloid runner can just be full of microbridge, due to the effect of surface tension, matrigel
Microbridge, a length of 400-1000 μm of the microbridge are not overflowed, width is 50-100 μm, and the spacing of two microbridges is 400-600 μm.
Preferably, cell culture unit and biogum are perfused between unit and unit and nutrition perfusion list is perfused in biogum
A length of 400-500 μm of microbridge between member, width are 50-60 μm, and the spacing of two microbridges is 400-500 μm.
Preferably, a length of 900-1000 μm of the microbridge between two adjacent groups cultivation platform, width are 50-80 μm, and spacing is
500-600μm。
In order to ensure that the nutritional ingredient in nutrient solution meets cell culture quantity, the Coculture chip packet enough
4-8 group cultivation platforms are included, every group of cultivation platform includes 3-10 cell culture unit, is passed through between flanking cell culture unit
Microbridge is connected to.Preferably, including four groups of cultivation platforms, it is in cross distribution, it is between four groups of cultivation platforms or relatively independent, or successively
All connections, or be connected to two-by-two;Every group of cultivation platform includes three cell culture units, is led between flanking cell culture unit
Cross microbridge connection
Compared with prior art, the advantages and positive effects of the present invention are:The present invention can be according to cultivation platform and cell
The quantity of unit is cultivated, various kinds of cell is formed and co-cultures, it is high-throughput, it can influence each other between real-time continuous observation various kinds of cell,
More true simulated in vivo environment.Whether it is connected to according between cultivation platform, several groups of co-cultivation platforms can be freely formed, according to
Different experiments demand carries out corresponding operating, realizes the function of kinds of experiments operation.It can be on the co-cultivation platform of various combination
Row control experiment facilitates observation experiment as a result, can avoid influence of the outside environmental elements to control experiment result.
Description of the drawings
Fig. 1 is the micro-structure schematic diagram that 12 kinds of cells co-culture chip in embodiment 1;
Fig. 2 is the micro-structure schematic diagram that 2 groups of 6 kinds of cells co-culture chip in embodiment 1;
Fig. 3 is the micro-structure schematic diagram that 4 groups of 3 kinds of cells co-culture chip in embodiment 1;
Fig. 4 is the micro-structure schematic diagram of 1 Single cell culture platform of embodiment;
Fig. 5 is single group nutrition perfusion cell schematics in Fig. 4;
Fig. 6 is multigroup nutrition perfusion cell schematics in Fig. 4;
Fig. 7 is that cell schematics are perfused in biogum in Fig. 4;
Fig. 8 is cell culture unit schematic diagram in Fig. 4;
Fig. 9 is that cell co-cultures chip schematic three dimensional views in embodiment 1;
It is marked in figure:1. nutrition perfusion unit, 1.1 inlets, 1.2 flow channel for liquids, 1.3 liquid cells, 1.4 go out liquid
Mouthful;2. unit, 2.1 glue-feeders, 2.2 colloid runners, 2.3 gum outlets are perfused in biogum;3. cell culture unit, 3.1 is thin
Born of the same parents' import, 3.2 cell runners, 3.3 cell culture chambers, 3.4 cell outlets;4. microbridge;5. into fluid column;6. vacuum suction fills
It sets;10. cultivation platform.
Specific implementation mode
In order to make the purpose , technical scheme and advantage of the present invention be clearer, below with reference to drawings and examples,
Invention is further described in detail.
The present embodiment proposes a kind of high-flux cell Combined culture chip, and various kinds of cell does not contact so that the life of cell
Long environment is and easy to operate closer to internal growing environment, is convenient for clinical application.
The Coculture chip, including shell, carrier and micro-structure;The shell is formed with carrier sealing-in, shell
Body material is dimethyl silicone polymer(PDMS), which has that plasticity is good, stable in physicochemical property, non-toxic and tasteless, transparent, logical
The good excellent performance of gas, suitable for the case material as the present invention.The thickness of the shell is 3-5mm.The carrier
Material is glass.
The micro-structure is located at shell inner surface, and the micro-structure is cavity, and the height of the cavity is 50-100 μm.
The micro-structure includes N groups cultivation platform 10, N >=1, the distribution of 10 rounded divergent shape of multigroup cultivation platform, N groups cultivation platform it
Between or relatively independent or every group between be sequentially communicated by microbridge 4, or selectivity two adjacent groups to adjacent(N-1)Between group
It is connected to by microbridge 4, such as can be adjacent two groups of connections or adjacent three groups of connections or adjacent(N-1)Group connection.
As shown in figure 4, every group of cultivation platform 10 includes three-decker, sequentially connected internal layer nutrition perfusion from inside to outside
Unit 2 and okioplast culture unit 3 is perfused in unit 1, middle layer biogum, is connected to by microbridge 4 between three;Every group of culture
Platform includes 1-10 cell culture unit 3, is connected to by microbridge between flanking cell culture unit.
As shown in figure 5, the internal layer nutrient solution perfusion unit 1 includes inlet 1.1, flow channel for liquids 1.2, liquid cell 1.3
With liquid outlet 1.4;In order to facilitate feed liquor and go out liquid, the inlet and liquid outlet both pass through shell and cavity are in communication with the outside;
The flow channel for liquids and liquid cell are between shell and carrier.The shape of the nutrient solution perfusion unit 1 is inside it
Cell culture unit and biogum perfusion unit be formed by depending on shape, shape sets requirement both can be cell culture
Room persistently provides nutrition again and can facilitate the connection between different tissues, and the liquid cell of the present embodiment uses triangular funne shape,
Multiple rounded divergent shape distributions of cultivation platform, i.e. the same center of circle is directed toward on the vertex of liquid cell in cultivation platform, such as Fig. 6 institutes
Show, be conducive to the area for increasing liquid cell, increase the storage volume of nutrient solution, nutrition is persistently provided for cell.
The inlet 1.1 is connect with into fluid column 5, by injecting nutrient solution in nutrient solution perfusion unit 1 into fluid column 5.
The liquid outlet 1.4 connects negative pressure suction device 6, starts the negative pressure suction device 6 when liquid filling and provides negative pressure so that into fluid column
Liquid in 5 starts to flow, and when liquid enters liquid outlet from inlet through flow channel for liquids, removes negative pressure suction device 6, at this time
Can continuous perfusion be achieved the purpose that by the gravity constant flow of itself liquid level difference into the liquid in fluid column.It is described negative
It can be vacuum suction micro syringe to press suction device.
Described into fluid column 5 is the narrow bottle of mouth under upper mouth width, and capacity is 200-300 μ L, and the lower port diameter of the bottle is
0.8-1mm, back cut diameter 4-6mm, bottle height are 40-45mm, into the liquid level of fluid column are 25-35mm when liquid filling.
The nutrient solution perfusion unit of the present embodiment, it is easy to operation, just only with negative pressure suction device and gravity
Can maintain perfusate the systems stay stablize perfusion, be not required to micro pump maintenance, reduce polluted in cell cultivation process it is several
Rate is suitble to the large-scale application of clinically Coculture.
As shown in fig. 6, the middle layer biogum perfusion unit 2 is located at internal layer nutrient solution perfusion unit 1 and okioplast
Between cultivating unit 3, there are glue-feeder 2.1, colloid runner 2.2 and gum outlet 2.3;On the colloid runner with the cell culture
The place of room contact is equipped with microbridge.The colloid runner 2.2 include main stem and(N-1)Branch, main stem form entire life
The frame of unit is perfused in object glue, and across entire cell culture unit, head end and end are separately connected glue-feeder 2.1 and gum outlet
2.3;Branch is between each two cell culture unit, for adjacent cell culture unit to be connected to by microbridge, head end
It is connected to main stem, end connects gum outlet 2.3.
Matrigel is perfused in the colloid runner;The matrigel is temperature-sensitive biochemical glue, is at 0-4 DEG C
Liquid condition is poured into biogum perfusion unit, so that it is full of colloid runner and each microbridge, 8- is maintained at 37 DEG C at this time
12h starts to be in gel, has certain intensity.The concentration of the matrigel and length of microbridge, width and colloid stream
The length in road, width design, in order to ensure that matrigel can just be full of microbridge, due to the effect of surface tension, matrigel
Microbridge is not overflowed.Therefore, a length of 400-1000 μm of microbridge is set, and width is 50-100 μm, and two microbridges are asked away from for 400-600
μm, the width of colloid runner is 150-250 μm, disclosure satisfy that requirement, it is preferred that unit is perfused with biogum in cell culture unit
Between and biogum perfusion unit and nutrition perfusion unit between microbridge is 400-500 μm a length of, width is 50-60 μm, spacing
It it is 400-500 μm, the width of colloid runner is 200-220 μm;The a length of 900-1000 of microbridge between two adjacent groups cultivation platform
μm, width is 50-80 μm, and spacing is 500-600 μm.Gelatinous matrigel can allow the small molecule in nutrient solution perfusion channel
Nutriment and chemotherapeutics pass through, without allowing cell to pass through, so as to fully ensure nutriment and chemotherapeutics more
It is dissipated in cell culture chamber;Cell factor can also be allowed to pass through, bioprotein, the small organic molecule generated such as cell metabolism
And inorganic matter so that the different cells in different cell culture rooms influence each other.
The okioplast culture unit 3 is located at 2 outside of middle layer biogum perfusion unit, as shown in figure 8, including cell
Import 3.1, one end closing, the other end connect cell culture chamber 3.3 by cell runner 3.2;The other end of the culturing room 3.3
Cell outlet 3.4 is connected by cell runner 3.2;Preferred culturing room 3.3 is rectangular.It is multiple thin in every group of cultivation platform 10
Born of the same parents' culture unit 3 is arranged in parallel, and the microbridge 4 passed through between flanking cell culturing room 3.3 on colloid runner is connected to.The biogum
Perfusion unit 2 surrounds each cell culture unit 3, to increase the contact area of culturing room 3.3 and nutrition perfusion unit 1.
The mixture of perfusion dilution glue and cell in the cell culture chamber 3.3, the dilution glue are using training completely
Nutrient solution obtains after matrigel is diluted 4-8 times, and the complete culture solution is that one kind in basic culture solution is added some and is rich in
The natural materials of amino acid, vitamin and base etc, that is, be added growth factor, and the various nutrition that can be used to meet microorganism lack
The growth of swaged bacterial strain needs.The dilution glue makes cell be in dimensional culture state, and cell is a concentration of in dilution glue
106-107/ml.Various kinds of cell can be cultivated in the multiple cell culture chamber, realize that the Combined culture of cell, different type are thin
Born of the same parents do not contact to each other, but its cell factor generated can be permeated by the matrigel in microbridge in whole system, be realized
Microenvironment simulation under closer to entity state so that cell is cultivated on chip closer to vivo environment.
Embodiment 1
Coculture chip as shown in Figs. 1-3 includes four groups of cultivation platforms, is in crossing distribution, and every group of culture is flat
Platform includes three cell culture units.Colloid runner 2.2 in every group of cultivation platform includes a main stem and Liang Tiao branches, two
Between three cell culture units, three cell culture units are connected to by microbridge respectively for branch.Cell in the present embodiment
It cultivates between unit and biogum perfusion unit and the microbridge a length of 400 between unit and nutrition perfusion unit is perfused in biogum
μm, width be 50 μm, spacing is 400 μm, the width of colloid runner is 200 μm;Microbridge between two adjacent groups cultivation platform is long
It it is 900 μm, width is 50 μm, and spacing is 500 μm.
It can also be sequentially communicated by microbridge between the liquid cell 1.3 of four groups of cultivation platforms, as shown in Figure 1, it can be achieved that 12
Kind of cell co-cultures, high-throughput, at the same observe 12 kinds it is intercellular influence each other, more true simulated in vivo environment.Four tissue cultures
It supports and can be two-by-two connected to by microbridge in platform, as shown in Figure 2, it can be achieved that 2 group of 6 class cell co-cultures.Four groups of cultivation platforms it
Between can be mutual indepedent, without connection, it can be achieved that 4 group of 3 class cell co-cultures between four groups of cultivation platforms as shown in Figure 3.Afterwards two
Kind situation can not only simulate human internal environment, while can also do check experiment on a chip, make one mesh of observation result
It is clear.
Above example is only several in the several preferred embodiment of the present invention, it is noted that the present invention is not limited to
Above-described embodiment;It for those of ordinary skill in the art, still can be to the technical solution recorded in previous embodiment
It modifies or equivalent replacement of some of the technical features;And these modifications or substitutions, do not make relevant art side
The essence of case is detached from the spirit and scope of claimed technical solution of the invention.